Nucleosome-specific T helper cells (Th cells) initiate and sustain the production of pathogenic, anti-nuclear autoantibodies during the onset and progression of systemic lupus erythematosus (SLE) through cognate interaction with autoimmune B cells (Mohan et al., 1993, J. Exp. Med. 177:1367; Desai-Mehta et al., 1995, J. Clin. Invest. 95:53; Kaliyaperumal et al., 1996, J. Exp. Med. 183:2459; Shi et al., 1998, J. Exp. Med. 187:367; Voll et al., 1997, Arthritis Rheum. 40:2162; Kretz-Rommel, et al., 1997, J. Clin. Invest. 99:1888). These SLE-associated Th cells are primarily responsible for driving the pathogenic autoimmune response. Without the help provided by these Th cells, autoimmune B cells are unable to produce the disease-causing (pathogenic) autoantibodies associated with SLE. Some of the critical epitopes (i.e. autoantigenic determinants) to which these Th cells are directed have been localized to the histone proteins of nucleosomes (i.e. DNA-protein complexes in the nuclei of animal cells). For example, in lupus prone mice, SLE-associated autoepitopes have been identified at amino acid positions 10-33 of the H2B histone protein, at amino acid positions 85-102 of the H3 histone protein, and at amino acid positions 16-39 and 71-94 of the H4 histone protein (Kaliyaperumal et al., 1996, J. Exp. Med. 183:2459).
SLE-associated autoimmune B cells recognize and bind autoantigenic determinants in DNA, histones, or other proteins in nucleosomes through surface molecules (i.e. B cell receptors, BCRs) which are antibody (i.e. immunoglobulin or Ig) receptors. Autoantigenic recognition and binding by an autoimmune B cell is followed by endocytosis and processing of the entire nucleosome particle which bears the recognized autoantigen. As illustrated in FIG. 1, the processed histone autoepitope (i.e. a histone peptide) is loaded onto a major histocompatibility complex class II molecule (i.e. a MHC class II molecule or I-Ad molecule) of the B cell, and is presented as a complex on the surface of the B cell in an interaction with an autoimmune Th cell which has receptors specific for nucleosomal histone peptide autoepitopes. Upon receiving this antigen-specific signal (i.e. signal 1) and other co-stimulatory signals (i.e. signal 2) from the B cell, the autoimmune Th cell helps the autoimmune B cell by recruiting intermolecular support and enabling the B cell to survive and differentiate into an autoantibody-producing B cell associated with SLE (Datta and Kaliyaperumal, 1997, Ann. New York Acad. Sci. 815:155; Datta, 2000, Nature Med. 6:259).
Autoimmune T cells of lupus-prone mice are spontaneously primed to SLE-associated autoepitopes early in life before overt autoantibody production or any clinical manifestations of the disease are present (Kaliyaperumal et al., 1996, J. Exp. Med. 183:2459). Moreover, immunization of pre-autoimmune mice with peptides corresponding to SLE-associated nucleosomal autoepitopes precipitates SLE-associated nephritis by triggering autoimmune T helper cells of subtype 1 (i.e. Th 1 cells) which, in turn, initiate anti-nuclear autoantibody production (Kaliyaperumal et al., 1996, J. Exp. Med. 183:2459). The T helper cell subtypes 2 and 0 (i.e., Th2 and Th0 cells, respectively) are also involved in the progression of SLE, as these T helper cell subtypes maintain autoantibody production (Mohan et al., 1993, J. Exp. Med. 177:1367; Nakajima et al., 1997, J. Immunol. 158:1466).
Unlike organ-specific autoimmune diseases in which the autoimmune response targets a restricted set of autoepitoes and is mediated by a single population of T cells, the autoimmune response in SLE involves a complex web of polyclonal T cell and B cell hyperactivity and appears to be directed by multiple susceptibility genes (Datta et al., 1982, J. Immunol. 129:1539; Klinman and Steinberg, 1987, J. Exp. Med. 165:1755; Cohen and Eisenberg, 1991, Ann. Rev. Immunol. 9:243; Chan and Shlomchik, 1998, J. Immunol. 160:51; Jongstra-Bilen, 1997, J. Immunol. 159:5810; Mohan et al., 1995, J. Immunol. 154:1470; Desai-Mehta, 1996, J. Clin. Invest. 97:2063; Koshy et al., 1996, J. Clin. Invest. 98:826; Liossis, et al., 1996, J. Clin. Invest. 98:2549; Wakeland et al., 1997, J. Clin. Immunol. 17:272; Vyse and Kotzin 1996, Opin. Immunol. 8:843; Kono and Theofilopoulos, 1996, J. Autoimmunity 9:437).
Previously, it has not been considered that a brief tolerogenic regimen of nucleosomal peptides could delay the development of SLE-associated nephritis. Moreover, chronic tolerogenic therapy with peptides has not previously been pursued, as it was not foreseen that such therapy might have the effect of prolonging survival and slowing SLE progression. There has long been a need in the art to identify immunololgically specific agents which could significantly affect the therapeutic outcome of SLE by delaying or preventing the onset and progression of the disease and complications thereof. The present invention provides a novel therapeutic approach to satisfying this need.
The present invention provides a composition comprising an isolated peptide which has an amino acid sequence corresponding to the amino acid sequence of a portion of a nucleosome histone protein and, and which is capable of promoting immunological tolerance in an animal having systemic lupus erythematosus.
The invention includes an isolated peptide which has an amino acid sequence corresponding to the amino acid sequence of a portion of a nucleosome histone protein, wherein the portion of the nucleosome histone protein corresponds to an autoepitope which is associated with systemic lupus erythematosus and which is recognized by one or more of an autoimmune T cell and an autoimmune B cell.
In one embodiment, the isolated peptide can correspond in amino acid sequence to a nucleosome histone protein which is selected from the group consisting of histone 1 (H1), histone 2A (H2A), histone 2B (H2B), histone 3 (H3), and histone 4 (H4).
In multiple embodiments, the isolated peptide comprises not more than 27 contiguous amino acids and has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26.
In other embodiments, the isolated peptide can further comprise a covalently attached moiety selected from the group consisting of a fluorophore, a chromophore, a biotin moiety, a light reactive group, and an enzyme cleavable group.
In one aspect, the invention includes a composition comprising a pharmaceutically acceptable carrier and an isolated peptide which has an amino acid sequence corresponding to the amino acid sequence of a portion of a nucleosome histone protein and, and which is capable of promoting immunological tolerance in an animal having systemic lupus erythematosus.
In another aspect, the invention includes an isolated nucleic acid encoding an isolated peptide which has an amino acid sequence corresponding to the amino acid sequence of a portion of a nucleosome histone protein and, and which is capable of promoting immunological tolerance in an animal having systemic lupus erythematosus. In this aspect, the isolated nucleic acid can comprise a nucleotide sequence selected from the group consisting of SEQ ID NOS.: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 and 52. Also included in this aspect of the invention is a vector comprising the isolated nucleic acid.
In multiple embodiments the isolated nucleic acid of the invention comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS.: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 and 52.
The invention also includes a cell comprising an isolated nucleic acid encoding an isolated peptide which has an amino acid sequence corresponding to the amino acid sequence of a portion of a nucleosome histone protein and, and which is capable of promoting immunological tolerance in an animal having systemic lupus erythematosus.
In multiple embodiments, the cell comprising the isolated nucleic acid is selected from the group consisting of a prokaryotic cell and a eukaryotic cell. In one embodiment, the cell is an insect cell.
The invention additionally includes a method of treating an animal having an autoimmune disorder. This method comprises administering to the animal an isolated peptide comprising a portion of a nucleosome histone protein, wherein the isolated peptide is capable of promoting immunological tolerance in an animal, thereby treating the autoimmune disorder in the animal.
In multiple embodiments, the autoimmune disorder is selected from the group consisting of rheumatoid arthritis, scleroderma, and systemic lupus erythematosus.
In other embodiments, the isolated peptide is administered in an amount which is from at least about 10 micrograms per kilogram of animal to at least about 1 gram per kilogram of animal, and from at least about 100 micrograms per kilogram of animal to about 600 micrograms per kilogram of animal.
In still other embodiments, the isolated peptide comprises not more than 27 contiguous amino acids and having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26.
Alternatively, the method of treating an animal having an autoimmune disorder comprises administering to the animal a modified histone peptide, wherein the modified histone peptide is an altered peptide ligand, and wherein the modified histone peptide is capable of promoting immunological tolerance in the animal, thereby treating the autoimmune disorder. In various embodiments of this method, the autoimmune disorder is selected from the group consisting of rheumatoid arthritis, scleroderma, and systemic lupus erythematosus, and the isolated peptide is administered in an amount which is from at least about 10 micrograms per kilogram of animal to at least about 1 gram per kilogram of animal, and from at least about 100 micrograms per kilogram of animal to about 600 micrograms per kilogram of animal.
In other embodiments, the modified peptide comprises not more than 27 contiguous amino acids and has an amino acid sequence which is at least one amino acid different relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26.
The invention includes a method of treating nephritis in an animal having systemic lupus erythematosus. This method comprises administering to the animal an isolated peptide comprising a portion of a nucleosome histone protein, wherein the isolated peptide is capable of promoting immunological tolerance in an animal, thereby alleviating the nephritis in the animal. Alternatively, this method comprises administering to the animal a modified histone peptide, wherein the modified histone peptide is an altered peptide ligand, and wherein the modified histone peptide is capable of promoting immunological tolerance in the animal, thereby alleviating the nephritis in the animal.
The invention encompasses a method of reducing the production of autoantibodies in an animal. This method comprises administering to the animal an isolated peptide comprising a portion of a nucleosome histone protein, wherein the isolated peptide is capable of promoting immunological tolerance in an animal, and wherein the peptide is administered in an amount sufficient to promote immunologic tolerance in the animal, thereby reducing the production of autoantibodies in the animal. Alternatively, this method comprises administering to said animal a modified histone peptide, wherein the modified histone peptide is an altered peptide ligand capable of promoting immunological tolerance in an animal, and wherein the modified histone peptide is administered in an amount sufficient to promote immunologic tolerance in the animal, thereby reducing the production of autoantibodies in the animal.
In another aspect, the invention encompasses a method of treating inflammation in an animal, which inflammation is caused by the production of autoantibodies in the animal. This method comprises administering to the animal an isolated peptide comprising a portion of a nucleosome histone protein, wherein the isolated peptide is capable of promoting immunological tolerance in an animal, and wherein the peptide is administered in an amount sufficient to promote immunological tolerance in the animal, thereby inhibiting the production of autoantibodies in the animal and alleviating inflammation in the animal. Alternatively, this method comprises administering to the animal a modified histone peptide, wherein the modified histone peptide is an altered peptide ligand capable of promoting immunological tolerance in an animal, and wherein the modified histone peptide is administered in an amount sufficient to promote immunological tolerance in the animal, thereby inhibiting the production of autoantibodies in the animal and alleviating inflammation in the animal.
In multiple embodiments, the invention provides a method of diagnosing systemic lupus erythematosus in an animal. This method comprises (a) contacting a sample from the animal with a composition comprising an isolated histone peptide complex, wherein said histone peptide complex comprises
i) a histone peptide portion, histone peptide portion comprising no more than 27 contiguous amino acids and having an amino acid sequence corresponding to a portion of a nucleosome histone protein;
ii) a fused portion having an amino acid sequence which corresponds to a portion of a protein selected from the group consisting of a major histocompatibility class II molecule and an immunoglobin; and compatible
iii) an indicator portion, wherein said indicator portion is a molecule which is capable of producing a detectable chemical signal, and which is selected from the group consisting of a flourophore, a chromophore, a light reactive moiety and a biotin moiety; and wherein each of said histone peptide portion, said fused portion, and said indication portion is covalently linked to at least one other component of the histone peptide complex; and (b) identifying in the animal the signal produced by the indicator portion, whereby the identification of the signal in the animal is an indication that the animal has systemic lupus erythematosus, thereby diagnosing systemic lupus erythematosus in the animal. In other embodiments, the invention includes a method of tracking an autoimmune cell associated with systemic lupus erythematosus in an animal. This method comprises
(a) contacting a sample from the animal with a composition comprising one or more of a modified histone peptide and an isolated histone peptide complex, wherein the modified histone peptide comprises
(i) an modified portion wherein said modified portion is a molecule which is capable of producing a detectable chemical signal, and which is selected from the group consisting of a flourophore, a chromophore, a light reactive moiety and a biotin moiety; and
(ii) a peptide portion comprising not more than 27 contiguous amino acids and
having an amino acid sequence corresponding to a nucleosome histone protein, and wherein the peptide portion and the modified portion are covalently linked; and wherein the histone peptide complex comprises
(iii) a histone peptide portion, comprising no more than 27 contiguous amino acids and having an amino acid sequence corresponding to a portion of a nucleosome histone protein,
(iv) a fused portion having an amino acid sequence which corresponds to a portion of a protein selected from the group consisting of a major histocompatibility class II molecule and an immunoglobin, and
(v) an indicator portion, wherein the indicator portion is a molecule which is capable of producing a detectable chemical signal, and which is selected from the group consisting of a flourophore, a chromophore, a light reactive moiety and a biotin moiety; and
wherein each of the histone peptide portion, the fused portion, and the indicator portion is covalently linked to at least one other component of the histone peptide complex; and
(b) identifying and monitoring in the animal the signal produced by the indicator portion, wherein the identification and monitoring of the signal in the animal constitutes the identification and monitoring of an autoimmune cell associated with systemic lupus erythematosus to which the histone protein complex is bound, thereby tracking the autoimmune cell associated with systemic lupus erythematosus in the animal.